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US8986769B2 - Methods for preserving endogenous TGF-β - Google Patents

Methods for preserving endogenous TGF-β Download PDF

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Publication number
US8986769B2
US8986769B2 US12/370,374 US37037409A US8986769B2 US 8986769 B2 US8986769 B2 US 8986769B2 US 37037409 A US37037409 A US 37037409A US 8986769 B2 US8986769 B2 US 8986769B2
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United States
Prior art keywords
tgf
whey protein
another embodiment
milk
present
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US12/370,374
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US20100104727A1 (en
Inventor
Juan M. Gonzalez
Dattatreya Banavara
John D. Alvey
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Mead Johnson Nutrition Co
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Mead Johnson Nutrition Co
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Priority to US12/370,374 priority Critical patent/US8986769B2/en
Assigned to MEAD JOHNSON NUTRITION COMPANY reassignment MEAD JOHNSON NUTRITION COMPANY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ALVEY, JOHN D., BANAVARA, DATTATREYA, GONZALEZ, JUAN M.
Priority to TW098135793A priority patent/TWI499379B/zh
Priority to MX2011003479A priority patent/MX2011003479A/es
Priority to BRPI0919758-3A priority patent/BRPI0919758A2/pt
Priority to EP09822753.1A priority patent/EP2337450B1/fr
Priority to CA2740295A priority patent/CA2740295C/fr
Priority to HK12102607.7A priority patent/HK1162122B/xx
Priority to PE2011000916A priority patent/PE20110847A1/es
Priority to NO09822753A priority patent/NO2337450T3/no
Priority to ES09822753.1T priority patent/ES2665933T3/es
Priority to RU2011120828/10A priority patent/RU2547590C2/ru
Priority to CZ20110238A priority patent/CZ2011238A3/cs
Priority to CN2009801418252A priority patent/CN102196723B/zh
Priority to PL09822753T priority patent/PL2337450T3/pl
Priority to PCT/US2009/061783 priority patent/WO2010048474A1/fr
Publication of US20100104727A1 publication Critical patent/US20100104727A1/en
Priority to CO11041153A priority patent/CO6361842A2/es
Publication of US8986769B2 publication Critical patent/US8986769B2/en
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    • A23L1/3056—
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A23L1/296—
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00—Processes
    • A23V2300/46—Ultra high pressure

Definitions

  • the present invention relates generally to methods of preserving the bioactivity of transforming growth factor- ⁇ (TGF- ⁇ ) in liquid nutritional products.
  • TGF- ⁇ transforming growth factor- ⁇
  • the present invention is directed, in an embodiment, to a method of preparing a liquid nutritional product that retains the bioactivity of TGF- ⁇ .
  • the steps involve selecting one or more protein ingredients that have been subjected to a heat load comprising medium-heat or less; combining the protein ingredients with all other components of the liquid nutritional product to form a slurry; subjecting the slurry to a pressure of from about 2500 per square inch (psi) to about 3500 psi at a temperature of from about 55° C. to about 65° C. for about 5 to about 20 seconds; subjecting the slurry to a temperature of from about 135° C. to about 150° C. for about 1.5 to about 15 seconds; and cooling the slurry to a temperature of less than about 8° C. over about 30 minutes or less.
  • psi per square inch
  • FIG. 1 illustrates an embodiment of a method of the present invention
  • FIG. 2 is a bar chart illustrating the bioactivity of infant formula produced according to the new process versus infant formula produced according to the standard process.
  • the present invention relates generally to methods of preserving the bioactivity of TGF- ⁇ in liquid nutritional products.
  • References related to such methods may include U.S. Pat. Nos. 7,057,016 or 6,057,430 to Cerletti.
  • TGF- ⁇ Transforming growth factor-beta
  • TGF- ⁇ is the general name for a family of polypeptides, the members of which have multifunctional regulatory activities.
  • Three differentially regulated mammalian isoforms (termed TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3) play important roles in a multitude of processes in the developing embryo, infant, child and adult.
  • TGF- ⁇ is a 25-kDa homodimeric cytokine known to mediate pleitropic functions both within the immune system and systemically.
  • TGF- ⁇ is expressed in several cell types in the intestinal mucosal including lymphocytes, epithelial cells, macrophages, and stromal cells as well as by T-cells, neutrophils, macrophages, epithelial cells, fibroblasts, platelets, osteoblasts, osteoclasts and others.
  • TGF- ⁇ is present in human breast milk and may influence multiple aspects of infant health and development.
  • TGF- ⁇ s are synthesized as large precursor proteins which consist of an amino-terminal pro-domain, comprising a signal sequence and latency-associated complex, and a mature carboxy-terminal subunit.
  • Biologically active TGF- ⁇ s are homodimers which consist of two identical, disulfide-linked mature subunits. Release of the TGF- ⁇ homodimer from the latency-associated complex is necessary for TGF- ⁇ to exert biological activity on target cells.
  • the nature of the latency-associated complex and the mechanisms responsible for TGF- ⁇ release are key to understanding TGF- ⁇ biological activity in vivo. In the human gut, this may be accomplished by the action of proteolytic enzymes, pH extremes, heat, calcium, and/or mechanical tearing.
  • TGF- ⁇ growth factor- ⁇
  • certain protein sources in nutritional products may provide a source of TGF- ⁇ .
  • the growth factor may be supplemented into the product. In either case, however, it is difficult to retain the biological activity of the TGF- ⁇ through the manufacturing process for a liquid nutritional product.
  • shelf stable, non-refrigerated liquid nutritional products are typically processed using a high heat treatment to destroy pathogenic bacterial spores.
  • a common retort process might heat the product to 130° C. for 2.5 minutes, depending upon the solids content of the nutritional product.
  • Ultra High Temperature (UHT) could be used. UHT heats the product to temperatures in the range of 135-150° C. for 1.5 to 15 seconds.
  • TGF- ⁇ a heat-labile bioactive compound, often cannot survive the common retort process required for manufacturing shelf-stable liquid nutritional products. These processes often significantly denature the native structure of TGF- ⁇ , thereby rendering it physiologically inert and unable to be activated in the human gut.
  • the technical problem to be solved by the present invention is to provide methods for preparing liquid nutritional products such that TGF- ⁇ and other heat-labile components retain their bioactivity and availability.
  • the present invention is directed to a processing method and a selection of ingredients that will retain TGF- ⁇ levels, bioactivity, and/or bioavailability in a shelf-stable, non-refrigerated, dairy-based liquid nutritional product.
  • dairy-based raw materials Prior to arrival at any processing plant, the manufacture of dairy-based raw materials, such as non-fat dry milk (also known as skim milk powder), whey protein powders, condensed milk, caseins, and others, requires some level or heating to ensure safety.
  • dairy-based raw materials are classified based on the cumulative heat treatments that they have received, as shown in Tables 1 and 2. Because it is difficult to determine the type of heat treatment the raw material has received upon inspection, the undenatured whey protein nitrogen (WPN) levels of the raw material are measured to convey such information.
  • Table 1 indicates the generally-accepted treatment parameters and WPN levels for non-fat dry milk.
  • the level of WPN in non-fat dry milk is the amount of undenatured whey protein present in the non-fat dry milk expressed in milligrams nitrogen per gram non-fat dry milk.
  • the milk powder may be rehydrated, heated in a saturated sodium chloride solution, and filtered.
  • the filtrate then contains the undenatured whey proteins and the non-protein nitrogen fractions.
  • the filtrate may then be diluted in a saturated salt solution and acidified, and the solution may be measured spectrophometrically.
  • the transmittance obtained may then be compared to a standard curve that plots the Kjeldahl nitrogen values from standards against transmittance for those standards. This method is often referred to as the Harland-Ashworth Test.
  • any method known in the art for determining WPN levels in non-fat dry milk may be used.
  • a “low-heat” non-fat dry milk is defined as one that contains at least about 6 mg/g WPN upon arrival for processing. Low-heat non-fat dry milk has likely experienced exposure to a temperature of less than about 70° C. for approximately 30 seconds to 5 minutes.
  • a “medium-heat” non-fat dry milk is defined as one that contains between about 1.51 mg/g and about 5.99 mg/g WPN upon arrival for processing.
  • Medium-heat non-fat dry milk has likely experienced exposure to a temperature in the range of about 70° C. to 78° C. for approximately 15 to 25 minutes.
  • a “high-heat” non-fat dry milk is defined as one that contains about 1.5 mg/g or less of WPN upon arrival for processing. High-heat non-fat dry milk has likely experienced exposure to a temperature of at least 88° C. for 30 minutes or more.
  • Table 2 indicates the treatment parameters and WPN levels for whey protein powders, including whey protein concentrate and whey protein isolate. See Mahmoud, et al., Factors Affecting Measurement of Undenatured Whey Protein Nitrogen in Dried Whey by a Modified Harland - Ashworth Test, J. Dairy Sci. 73:1694-1699 (1990).
  • the WPN levels in whey protein powders are higher than those of standard non-fat dry milk because the whey protein composition in whey protein powder, such as whey protein concentrate, which is about 50% protein, is at least 6 to 7 times higher than in nonfat dry milk on a dry basis. As the method detects denaturation in whey protein, results of whey protein powders are expected to be higher than results obtained with non-fat dry milk.
  • a “low-heat” whey protein powder is defined as one that contains at least about 14.5 mg/g WPN upon arrival for processing. Low-heat whey protein powder has likely experienced exposure to a temperature of less than about 63° C. for approximately 30 seconds to 30 minutes.
  • a “medium-heat” whey protein powder is defined as one that contains between about 5.99 mg/g and about 14.49 mg/g WPN upon arrival for processing.
  • Medium-heat whey protein powder has likely experienced exposure to a temperature in the range of about 70° C. to 78° C. for approximately 15 to 25 minutes.
  • a “high-heat” whey protein powder is defined as one that contains about 6.0 mg/g or less of WPN upon arrival for processing. High-heat whey protein powder has likely experienced exposure to a temperature of at least 91° C. for 30 minutes or more.
  • the selection of ingredients according to the invention is important because the levels of heat treatment impact the physicochemical properties of a protein source as well as its value in the processing of foods.
  • the heat-induced denaturation of whey proteins causes them to interact with the surface of casein micelles. This denaturation provides higher heat stability to milk casein during liquid formula manufacture. However, this process also denatures a number of bioactive components present in milk, such as growth factors like TGF- ⁇ .
  • the method comprises the particular selection of the protein material according to the heat load received during its manufacture and the thermal processes applied to the nutritional product during processing.
  • the selection of protein materials for the nutritional product includes a source of non-fat dry milk.
  • the non-fat dry milk source may have a WPN of greater than about 1.5 mg/g.
  • the non-fat dry milk source may have a WPN of greater than about 2.0 mg/g.
  • the non-fat dry milk source may have a WPN of greater than about 3.0 mg/g.
  • the non-fat dry milk source may have a WPN of greater than about 4.0 mg/g.
  • the non-fat dry milk source may have a WPN of greater than about 5.0 mg/g.
  • the non-fat dry milk source may have a WPN of greater than about 6.0 mg/g.
  • the non-fat dry milk source may have a WPN of from about 3 mg/g to about 10 mg/g. In some embodiments, the non-fat dry milk contains a WPN level of about 1.5 mg/g to about 8 mg/g. In still other embodiments, the non-fat dry milk contains a WPN level of between about 5 mg/g and about 8 mg/g. While the WPN levels are classified herein according to low-, medium-, and high-heat, it is to be understood that a medium-heat non-fat dry milk having a WPN level closer to about 5.99 mg/g than to about 1.5 mg/g may have greater endogenous quantities of TGF- ⁇ present therein.
  • the selection of protein materials for the nutritional product includes a source of whey protein powder.
  • the whey protein powder may be selected from the group consisting of sweet whey, demineralized whey, whey protein concentrate, whey protein isolate, and combinations thereof.
  • the whey protein powder source has received medium-heat, low-heat, or no heat.
  • the whey protein powder may have a WPN of greater than about 6.0 mg/g.
  • the whey protein powder may have a WPN of greater than about 8.0 mg/g.
  • the whey protein powder may have a WPN of greater than about 10.0 mg/g.
  • the whey protein powder may have a WPN of greater than about 12.0 mg/g. In a still further embodiment, the whey protein powder may have a WPN of greater than about 14.0 mg/g. In a particular embodiment, the whey protein powder may have a WPN of greater than about 14.5 mg/g.
  • the whey protein powder may have a WPN of from about 6 mg/g to about 15 mg/g. In some embodiments, the whey protein powder contains a WPN level of about 10 mg/g to about 20 mg/g. In still other embodiments, whey protein powder contains a WPN level of between about 12 mg/g and about 15 mg/g. While the WPN levels are classified herein according to low-, medium-, and high-heat, it is to be understood that a medium-heat whey protein powder having a WPN level closer to about 14.5 mg/g than to about 6.0 mg/g may have greater endogenous quantities of TGF- ⁇ present therein.
  • the selection of protein materials for the nutritional product includes a source of casein selected from the group consisting of whole milk powder, milk protein concentrate, milk protein isolate, and combinations thereof.
  • the casein source has been selected based upon a minimal heat treatment history.
  • both casein and whey protein sources are utilized in the nutritional product.
  • protein ingredients are selected based upon their manufacturing heat treatment and combined with all other components of the liquid nutritional product to form a slurry.
  • the slurry is first pasteurized.
  • the pasteurization may be conducted at a temperature of about 70° C. to about 75° C. for approximately 5 to 25 seconds.
  • the pasteurization may be conducted at a temperature of about 72° C. for approximately 10-20 seconds.
  • the pasteurization may be conducted at a temperature of about 72° C. for approximately 15 seconds.
  • the homogenization step may be conducted at a pressure of between about 2500 pounds per square inch (psi) and 3500 psi. In other embodiments, the pressure may be about 3000 psi.
  • the temperature range may be from about 55° C. to about 65° C. for the homogenization step.
  • the homogenization step in an embodiment, may take place for about 5 to 20 seconds. In a particular embodiment, the homogenization step is conducted at a pressure of about 3000 psi and a temperature in the range of about 55° C. to about 65° C. for about 5 to 20 seconds.
  • the homogenized emulsion is then UHT-treated.
  • the UHT treatment is conducted at a temperature in the range of about 135° C. to about 150° C. In other embodiments, the UHT treatment is conducted at a temperature in the range of about 141° C. to about 145° C. The UHT treatment may take place for about 1.5 to 15 seconds. In a particular embodiment, the UHT treatment is conducted at a temperature in the range of about 141° C. to about 145° C. for approximately 5 seconds.
  • the process then involves a rapid cooling step.
  • the product may be cooled to a temperature of less than about 8° C.
  • the cooling step may occur in less than about 30 minutes.
  • the product After the product is cooled, the product may be aseptically filled in the desired format to render a shelf-stable, non-refrigerated dairy product having active levels of TGF- ⁇ therein.
  • the slurry may be processed using high-pressure processing technology, under a combination of pressure in the range of about 400 MPa to about 600 MPa and a temperature in the range of about of 60° C. to about 80° C. for about 1 to 3 minutes.
  • This high-pressure processed material can then be homogenized and even further UHT treated, if needed, as discussed above.
  • This high-pressure alternate embodiment allows the nutritional product to retain its TGF- ⁇ bioactivity.
  • the high-pressure treated material can also be combined with one or all of the above processes to obtain a shelf-stable product which has active TGF- ⁇ and other bioactive compounds.
  • the inventive process is used to produce shelf-stable, non-refrigerated liquid products.
  • the process may also be used to produce refrigerated products, however.
  • the method can be modified in order to supplement the endogenous levels of TGF- ⁇ present in the selected raw materials.
  • the nutritional product may be an infant formula.
  • infant means a person not more than 12 months of age.
  • infant formula applies to a composition in liquid or powdered form intended for use, where necessary, as a substitute for human milk (breast milk substitute) in meeting the normal nutritional requirements of infants.
  • the nutritional product may be a human milk fortifier, meaning it is a composition which is added to human milk in order to enhance the nutritional value of human milk.
  • the inventive composition may be in powder or liquid form.
  • the inventive nutritional product may be a follow-up formula.
  • the term “follow-up formula” as used herein refers to foods intended for use as a liquid part of the weaning diet for the infant from the 6 th month of life on and for young children.
  • the term “young child” or “young children” means persons from the age of more than 12 months up to the age of three years.
  • the inventive nutritional product may be a children's nutritional composition.
  • the term “child” or “children” as used herein means persons over the age of 3 years and prior to adolescence.
  • the inventive nutritional product may be a growing-up milk.
  • growing-up milk refers to a broad category of milk-based fortified beverages intended to be used as a part of a diverse diet in order to support the normal growth and development of children from the ages of 1 to 6 years.
  • the composition is an acidified product.
  • the term “acidified product” refers to a nutritional composition which has a finished equilibrium pH of 4.6 or below and a water activity greater than 0.85.
  • the nutritional product may be a medical food.
  • medical food is defined as a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.
  • a product In general, to be considered a medical food, a product must, at a minimum, meet the following criteria: the product must be a food for oral or tube feeding; the product must be labeled for the dietary management of a specific medical disorder, disease or condition for which there are distinctive nutritional requirements; and the product must be intended to be used under medical supervision.
  • the nutritional products of the invention may provide minimal, partial, or total nutritional support.
  • the compositions may be nutritional supplements or meal replacements.
  • the compositions may be administered in conjunction with a food or nutritional composition.
  • the compositions can either be intermixed with the food or other nutritional compositions prior to ingestion by the subject or can be administered to the subject either before or after ingestion of a food or nutritional composition.
  • the compositions may be administered to preterm infants receiving infant formula, breast milk, a human milk fortifier, or combinations thereof.
  • compositions may, but need not, be nutritionally complete.
  • nutritionally complete means that the nutritional composition of the present invention provides adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for normal growth.
  • essential refers to any nutrient which cannot be synthesized by the body in amounts sufficient for normal growth and to maintain health and which therefore must be supplied by the diet.
  • conditionally essential as applied to nutrients means that the nutrient must be supplied by the diet under conditions when adequate amounts of the precursor compound is unavailable to the body for endogenous synthesis to occur.
  • composition which is “nutritionally complete” for the preterm infant will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the preterm infant.
  • the composition which is “nutritionally complete” for the term infant will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the term infant.
  • composition which is “nutritionally complete” for a child will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of a child.
  • the nutritional product is an infant formula or human milk supplement, it may be a product for a full-term infant, a preterm infant, a low-birth-weight infant, a very-low-birth-weight infant, or an extremely low birth weight infant.
  • full-term refers to neonatal infants born after about 37 weeks of gestation through 42 weeks gestation but less than 1 month of age.
  • full-term infant or infant refers to an infant less than twelve months of age.
  • preterm or “preterm infant” includes infants born prior to about 37 weeks of gestation.
  • low birth weight or “low birth weight infant” are those infants are those weighing from about 3.3 to about 5.5 pounds at birth. “Very-low-birth-weight infants” are those weighing less than about 3.3 to about 2.2 pounds at birth. “Extremely low birth weight” or “extremely low birth weight infants” are those weighing less than 2.2 pounds at birth.
  • the nutritional product formed via the method of the invention may be administered enterally or parenterally.
  • enterally means through or within the gastrointestinal, or digestive, tract
  • enteral administration includes oral feeding, intragastric feeding, transpyloric administration, or any other introduction into the digestive tract.
  • parenterally means taken into the body or administered in a manner other than through the digestive tract, such as by intravenous or intramuscular injection.
  • Bovine milk protein sources useful in practicing the present invention include, but are not limited to, milk protein powders, milk protein concentrates, milk protein isolates, nonfat milk solids, nonfat milk, nonfat dry milk, whey protein, whey protein isolates, whey protein concentrates, sweet whey, acid whey, casein, acid casein, caseinate (e.g. sodium caseinate, sodium calcium caseinate, calcium caseinate) and any combinations thereof.
  • the composition of the invention may comprise a children's nutritional composition provided as a growing-up milk.
  • Such invention may have a standard serving size of 200 ml, providing from about 60 to 75 kcal/100 ml of energy, with a recommended intake of two to three servings per day.
  • the amounts and types of proteins, lipids and carbohydrates may vary. Protein may comprise from about 2.5 to 3.75 g/100 kcal, with carbohydrate providing from about 11 to about 16.5 g/100 kcal and lipids comprising from about 2.2 to about 4.4 g/100 kcal.
  • Carbohydrate sources may be any known or used in the art as suitable for nutritional compositions, including but not limited to those disclosed herein.
  • Vitamin C When Vitamin C is present in the growing-up milk, it may be present in the range of about 0.1 to about 10 mg/serving. In another embodiment, vitamin C may be present at the level of 5 mg/serving. Any source of vitamin C known in the art to have nutritional uses, including, but not limited to those previously disclosed for practicing the invention, may be suitable for use in the present composition. In an embodiment, sources of vitamin C for use in the growing-up milk include L-ascorbic acid, sodium L-ascorbate, calcium L-ascorbate, ascorbyl palmitate, and any combinations thereof.
  • thiamin When thiamin is present in the growing-up milk, it may be present in the range of about 0.01 to about 0.5 mg/serving. In another embodiment, thiamin may be present in the range of 0.05 to about 0.15 mg/serving. In yet another embodiment, thiamin may be in the range of 0.08 to 0.10 mg/serving. Any source of thiamin known in the art to have nutritional uses, including, but not limited to those previously disclosed for practicing the invention, may be suitable for use in the present composition. In an embodiment, sources of thiamin for use in the growing-up milk include thiamin hydrochloride, thiamin mononitrate and any combinations thereof.
  • riboflavin When riboflavin is present in the growing-up milk, it may be present in the range of about 0.01 to about 0.5 mg/serving. In another embodiment, riboflavin may be present in the range of 0.05 to about 0.15 mg/serving. In yet another embodiment, riboflavin may be in the range of 0.08 to 0.10 mg/serving. Any source of riboflavin known in the art to have nutritional uses, including, but not limited to those previously disclosed for practicing the invention, may be suitable for use in the present composition. In an embodiment, sources of riboflavin for use in the growing-up milk include free riboflavin, sodium riboflavin, riboflavin-5′phosphate, and any combinations thereof.
  • vitamin B 6 When vitamin B 6 is present in the growing-up milk, it may be present in the range of about 0.01 to about 0.5 mg/serving. In another embodiment, vitamin B 6 may be present in the range of 0.05 to about 0.15 mg/serving. In yet another embodiment, the level of vitamin B 6 may be in the range of 0.08 to 0.10 mg/serving. Any source of vitamin B 6 known in the art to have nutritional uses, including, but not limited to those previously disclosed for practicing the invention, may be suitable for use in the present composition. In an embodiment, sources of vitamin B 6 for use in the growing-up milk include pyridoxine hydrochloride, pyridoxine-5′-phosphate and any combinations thereof.
  • folate When folate is present in the growing-up milk, it may be present in the range of 5 to 50 mcg/serving. In another embodiment, the folate content may be 10 to 40 mcg/serving. In yet another embodiment, the folate content may be within the range of 20 to 35 mcg/serving. Any source of folate known in the art to have nutritional uses, including, but not limited to those previously disclosed for practicing the invention, may be suitable for use in the present composition. In an embodiment, the source of folate for use in the growing-up milk is folic acid.
  • vitamin D When vitamin D is present in the growing-up milk, it may be present in the range of 0.1 to about 2 mcg/serving. In yet another embodiment, the vitamin D content of the growing-up milk may be 0.5 to 1 mcg/serving. Any source of vitamin D known in the art to have nutritional uses, including, but not limited to those previously disclosed for practicing the present invention, may be suitable for use in the present composition. In an embodiment, sources of vitamin D for use in the growing-up milk include cholecalciferol, ergocalciferol and any combinations thereof.
  • the inventive composition is a growing-up milk formulated for children between the ages of 1 to 6 years
  • vitamins and minerals may be added in varying amounts and ranges based on a per-serving basis.
  • one serving of the growing-up milk may contain from about 15% to about 50% of the Estimated Average Requirement (EAR) for children between the ages of 1 and 6 years for the following nutrients: vitamin E, vitamin K, niacin, pantothenic acid, vitamin B 12 , biotin, choline, potassium, magnesium, phosphorus, chloride, copper, selenium, fluoride, and any combinations thereof.
  • EAR Estimated Average Requirement
  • one serving of the growing-up milk may contain from about 20% to about 30% of the EAR for children between the ages of 1 and 6 years for the following nutrients: vitamin E, vitamin K, niacin, pantothenic acid, vitamin B 12 , biotin, choline, potassium, magnesium, phosphorus, chloride, copper, selenium, fluoride, and any combinations thereof. Any known sources of these nutrients having nutritional uses, including, but not limited to those disclosed herein may be suitable for use in the composition.
  • composition of the invention may optionally contain other substances that may have a beneficial effect on the host such as lactoferrin, nucleotides, nucleosides, immunoglobulins, CMP equivalents (cytidine 5′-monophosphate, free acid), UMP equivalents (uridine 5′-monophosphate, disodium salt), AMP equivalents (adenosine 5′-monophosphate, free acid), GMP equivalents (guanosine 5′-monophosphate, disodium salt), and combinations thereof.
  • CMP equivalents cytidine 5′-monophosphate, free acid
  • UMP equivalents uridine 5′-monophosphate, disodium salt
  • AMP equivalents adenosine 5′-monophosphate, free acid
  • GMP equivalents guanosine 5′-monophosphate, disodium salt
  • the nutritional composition may contain one or more probiotics.
  • probiotic means a microorganism that exerts beneficial effects on the health of the host. Any probiotic known in the art may be acceptable in this embodiment provided it achieves the intended result.
  • the probiotic may be selected from Lactobacillus species, Lactobacillus rhamnosus GG, Bifidobacterium species, Bifidobacterium longum, and Bifidobacterium animalis subsp. lactis BB-12.
  • the amount of polydextrose may be about 0.3 mg/100 Kcal.
  • galacto-oligosaccharide and polydextrose are supplemented into the nutritional composition in a total amount of about 0.6 mg/100 Kcal.
  • the amount of galacto-oligosaccharide may be about 0.3 mg/100 Kcal and the amount of polydextrose may be about 0.3 mg/100 Kcal.
  • the nutritional composition is supplemented with both DHA and ARA.
  • the weight ratio of ARA:DHA may be from about 1:3 to about 9:1. In one embodiment of the present invention, this ratio is from about 1:2 to about 4:1. In yet another embodiment, the ratio is from about 2:3 to about 2:1. In one particular embodiment the ratio is about 2:1. In another particular embodiment of the invention, the ratio is about 1:1.5. In other embodiments, the ratio is about 1:1.3. In still other embodiments, the ratio is about 1:1.9. In a particular embodiment, the ratio is about 1.5:1. In a further embodiment, the ratio is about 1.47:1.
  • the level of DHA is in the range of about 0.0% and 1.00% of fatty acids, by weight.
  • the level of DHA may be about 0.32% by weight. In some embodiments, the level of DHA may be about 0.33% by weight. In another embodiment, the level of DHA may be about 0.64% by weight. In another embodiment, the level of DHA may be about 0.67% by weight. In yet another embodiment, the level of DHA may be about 0.96% by weight. In a further embodiment, the level of DHA may be about 1.00% by weight.
  • the amount of DHA in an embodiment of the present invention is typically from about 3 mg per kg of body weight per day to about 150 mg per kg of body weight per day. In one embodiment of the invention, the amount is from about 6 mg per kg of body weight per day to about 100 mg per kg of body weight per day. In another embodiment the amount is from about 10 mg per kg of body weight per day to about 60 mg per kg of body weight per day. In yet another embodiment the amount is from about 15 mg per kg of body weight per day to about 30 mg per kg of body weight per day.
  • the amount of DHA in the nutritional composition may vary from about 5 mg/100 kcal to about 80 mg/100 kcal. In one embodiment of the present invention, DHA varies from about 10 mg/100 kcal to about 50 mg/100 kcal; and in another embodiment, from about 15 mg/100 kcal to about 20 mg/100 kcal. In a particular embodiment of the present invention, the amount of DHA is about 17 mg/100 kcal.
  • the amount of ARA in the nutritional composition may vary from about 10 mg/100 kcal to about 100 mg/100 kcal. In one embodiment of the present invention, the amount of ARA varies from about 15 mg/100 kcal to about 70 mg/100 kcal. In another embodiment, the amount of ARA varies from about 20 mg/100 kcal to about 40 mg/100 kcal. In a particular embodiment of the present invention, the amount of ARA is about 25 mg/100 kcal.
  • the nutritional composition may be supplemented with oils containing DHA and ARA using standard techniques known in the art.
  • DHA and ARA may be added to the formula by replacing an equivalent amount of an oil, such as high oleic sunflower oil, normally present in the formula.
  • the oils containing DHA and ARA may be added to the formula by replacing an equivalent amount of the rest of the overall fat blend normally present in the formula without DHA and ARA.
  • a LCPUFA source which contains EPA is used in the nutritional composition.
  • a LCPUFA source which is substantially free of EPA is used in the nutritional composition.
  • the nutritional composition contains less than about 16 mg EPA/100 kcal.
  • the nutritional composition contains less than about 10 mg EPA/100 kcal.
  • the nutritional composition contains less than about 5 mg EPA/100 kcal.
  • Another embodiment of the invention includes a nutritional composition that is free of even trace amounts of EPA.
  • the nutritional composition of the invention also may contain emulsifiers.
  • suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soy), and/or mono- and di-glycerides and mixtures thereof.
  • lecithin e.g., from egg or soy
  • mono- and di-glycerides and mixtures thereof e.g., from egg or soy
  • Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product.
  • the nutritional composition of the invention may optionally contain one or more stabilizers.
  • Suitable stabilizers for use in the nutritional composition of the present invention include, but are not limited to, gum Arabic, gum ghatti, gum karaya, gum tragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum, pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC (sodium carboxymethylcellulose), methylcellulose hydroxypropyl methylcellulose, hydroxypropyl cellulose, DATEM (diactyl tartaric acid esters of mono- and di-glycerides), dextran, carrageenans, and mixtures thereof.
  • the nutritional composition of the present invention may optionally include one or more preservatives that may also be added to extend product shelf life.
  • Suitable preservatives include, but are not limited to, potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate, calcium disodium EDTA, and mixtures thereof.
  • the level of TGF- ⁇ in the inventive composition is from about 0.0150 (pg/ ⁇ g) ppm to about 0.1000 (pg/ ⁇ g) ppm. In another embodiment, the level of TGF- ⁇ in the inventive composition is from about 0.0225 (pg/ ⁇ g) ppm to about 0.0750 (pg/ ⁇ g) ppm. In yet another embodiment, the level of TGF- ⁇ in the inventive composition is from about 0.0300 (pg/ ⁇ g) ppm to about 0.0600 (pg/ ⁇ g) ppm. In a particular embodiment, the level of TGF- ⁇ in the inventive composition is about 0.0340 (pg/ ⁇ g) ppm.
  • the level of TGF- ⁇ in the inventive composition is from about 2500 pg/mL to about 10,000 pg/mL composition. In another embodiment, the level of TGF- ⁇ in the inventive composition is from about 3000 pg/mL to about 8000 pg/mL. In yet another embodiment, the level of TGF- ⁇ in the inventive composition is from about 4000 pg/mL to about 6000 pg/mL. In a particular embodiment, the level of TGF- ⁇ in the inventive composition is about 5000 pg/mL.
  • the level of TGF- ⁇ 1 in the inventive composition is from about 0.0001 (pg/ ⁇ g) ppm to about 0.0075 (pg/ ⁇ g) ppm. In another embodiment, the level of TGF- ⁇ 1 in the inventive composition is from about 0.0010 (pg/ ⁇ g) ppm to about 0.0050 (pg/ ⁇ g) ppm. In yet another embodiment, the level of TGF- ⁇ 1 in the inventive composition is from about 0.0020 (pg/ ⁇ g) ppm to about 0.0035 (pg/ ⁇ g) ppm. In still another embodiment, the level of TGF- ⁇ 1 in the inventive composition is about 0.0030 (pg/ ⁇ g) ppm.
  • the ratio of TGF- ⁇ 1:TGF- ⁇ 2 in the inventive composition is in the range of about 1:1 to about 1:20. In another embodiment, the ratio of TGF- ⁇ 1:TGF- ⁇ 2 in the inventive composition is in the range of about 1:5 to about 1:15. In still another embodiment, the ratio of TGF- ⁇ 1:TGF- ⁇ 2 in the inventive composition is in the range of about 1:8 to about 1:13. In a particular embodiment, the ratio of TGF- ⁇ 1:TGF- ⁇ 2 in the inventive composition is about 1:11.
  • the bioactivity of TGF- ⁇ within the inventive composition is from about 500 ng Eq/100 kcal to about 5000 ng Eq/100 kcal. In another embodiment, the bioactivity of TGF- ⁇ within the inventive composition is from about 750 ng Eq/100 kcal to about 3000 ng Eq/100 kcal. In yet another embodiment, the bioactivity of TGF- ⁇ within the inventive composition is from about 800 ng Eq/100 kcal to about 2500 ng Eq/100 kcal. In one embodiment, the bioactivity is about 860 ng Eq/100 kcal. In another embodiment, the bioactivity is about 1700 ng Eq/100 kcal. In another embodiment, the bioactivity is about 1200 ng Eq/100 kcal.
  • the bioactivity of TGF- ⁇ in the inventive composition can be defined in terms of IC 50 in a HT-2 cell growth inhibition assay.
  • the bioactivity of the composition comprises an IC 50 from about 1.1 mg/ml to about 5.0 mg/ml.
  • the bioactivity of the composition comprises an IC 50 from about 1.2 mg/ml to about 3.0 mg/ml.
  • the bioactivity of the composition comprises an IC 50 from about 1.3 mg/ml to about 3.0 mg/ml.
  • the bioactivity of the composition comprises an IC 50 from about 1.3 mg/ml to about 2.0 mg/ml.
  • the bioactivity of the composition comprises an IC 50 of about 1.5 mg/ml.
  • This example illustrates an embodiment of the method of the invention.
  • the ingredients set forth in Table 3 were intermixed.
  • the mixture was then subjected to direct steam injection at 73° C. for 15 to 30 seconds. Afterward, the mixture was homogenized at 55° C. to 65° C. for 5 to 15 seconds. Finally, the mixture was retorted at 141° C. to 145° C. for 3 to 5 seconds. The mixture was then aseptically packaged and sealed.
  • This example illustrates an embodiment of a powdered infant formula of the present invention.
  • the infant formula described in this example contains approximately 4 g/L of galacto-oligosaccharide and has an ARA level of 25 mg/100 kcal.
  • the formula contains 5.6 g fat/100 kcal, to achieve a fat content which is similar to human milk.
  • the formula additionally has a low buffer strength.

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US12/370,374 US8986769B2 (en) 2008-10-24 2009-02-12 Methods for preserving endogenous TGF-β
TW098135793A TWI499379B (zh) 2008-10-24 2009-10-22 保存內源性轉型生長因子－β（ＴＧＦ－β）的方法
NO09822753A NO2337450T3 (fr) 2008-10-24 2009-10-23
CZ20110238A CZ2011238A3 (cs) 2008-10-24 2009-10-23 Metody pro zachování endogenního TGF-beta
EP09822753.1A EP2337450B1 (fr) 2008-10-24 2009-10-23 Procédés de conservation de tgf-bêta endogène
CA2740295A CA2740295C (fr) 2008-10-24 2009-10-23 Procedes de conservation de tgf-beta endogene
HK12102607.7A HK1162122B (en) 2008-10-24 2009-10-23 Methods for preserving endogenous tgf-beta
PE2011000916A PE20110847A1 (es) 2008-10-24 2009-10-23 Procedimiento de preparacion de un producto proteico nutricional liquido que conserva la bioactividad de tgf-beta
MX2011003479A MX2011003479A (es) 2008-10-24 2009-10-23 Metodos para conservar endogenos del factor de crecimiento-beta.
ES09822753.1T ES2665933T3 (es) 2008-10-24 2009-10-23 Métodos para preservar TGF- endógeno
RU2011120828/10A RU2547590C2 (ru) 2008-10-24 2009-10-23 Способы сохранения эндогенного tgf-бета
BRPI0919758-3A BRPI0919758A2 (pt) 2008-10-24 2009-10-23 Métodos de preservação de tgf-beta endógeno
CN2009801418252A CN102196723B (zh) 2008-10-24 2009-10-23 保存内源TGF-β的方法
PL09822753T PL2337450T3 (pl) 2008-10-24 2009-10-23 Sposoby zachowywania endogennego tgf-beta
PCT/US2009/061783 WO2010048474A1 (fr) 2008-10-24 2009-10-23 Procédés de conservation de tgf-bêta endogène
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